Modern healthcare relies extensively on a range of chemical and biochemical analytical tests on a variety of body fluids to enable diagnosis, therapy and management of disease. Medical and technological advances have considerably expanded the scope of diagnostic testing over the past few decades. Moreover, an increasing understanding of the human body, together with the emergence of technologies, such as microsystems and nanotechnology, are expected to have a profound impact on diagnostic technology.
Increasingly, diagnostic tests in hospitals are carried out at the point-of-care (PoC), in particular, in situations where a rapid response is a prime consideration and therapeutic decisions have to be made quickly. Despite recent advances in PoC testing, several compelling needs remain unmet. In particular, many assay methods require extensive pre-treatment of a complex sample matrix (such as whole blood) prior to analysis. For example, many sample preparation methods involve the disruption (lysis) and/or removal (filtration/centrifugation) of blood cells. Consequently, these steps are usually performed manually by highly skilled laboratory personnel. This requirement precludes the use of such assays in a PoC setting. There is therefore a strong unmet need to automate whole blood sample preparation methods. The detection and measurement of analyte levels in biological media (e.g. blood, plasma, saliva, urine) is often difficult due to the association of the analyte with components of the sample matrix (e.g. plasma proteins and lipid membranes). The free (unbound) molecule concentration (which can be in the picomolar range) is often below the sensitivity limits of the most commonly used measurement techniques (e.g. electrochemical, optical). Furthermore, several endogenous interfering compounds may be present and often need to be removed before analysis. To avoid these problems, state of the art methods for analyte measurement in biological samples usually incorporate one or more sample preparation steps that are intended to alter the composition of the sample being tested to make it compatible with the requirements of the detection system being employed (using e.g. optical, electrochemical, mass measurement techniques). Sample preparation methods can include, but are not limited to; antibody labelling, cell disruption (lysis) by chemical, mechanical or osmotic means, metered dilution into an alternative solvent, solid phase extraction, filtration, centrifugation and/or evaporation.
There are numerous examples in the prior art of assays that employ automated sample preparation from whole blood, including lateral flow immunoassays for plasma/cell separation, as for instance disclosed in U.S. Pat. No. 5,906,742, optical haemoglobin assays as for instance disclosed in U.S. Pat. No. 6,831,733 and haematology analysers utilizing cell lysis as for instance disclosed in U.S. Pat. No. 7,247,484.
However, many other biological assays require extensive manual sample preparation steps prior to analysis. For example, an established “gold standard” HPLC assay for the measurement of the anaesthetic drug propofol in whole blood usually involves dilution of a whole blood sample into a known volume of organic solvent, followed by centrifugation and injection of a known volume of the supernatant into an HPLC device for analysis, as disclosed by G. F. Plummer et al. in Improved method for the determination of propofol in blood by high-performance liquid chromatography with fluorescence detection,” Journal of Chromatography, vol. 421, no. 1, p. 171, 1987 and by R. A. Uebel et al. in “Electrochemical determination of 2,6-diisopropylphenol after high-performance liquid chromatography of extracts from serum,” Journal of Chromatography, vol. 526, no. 1, pp. 293-295, March 1990.
To date, no method for automating these sample preparation steps has been established.
The complex and time-consuming nature of the sample preparation steps involved in many biological assays mean that they are routinely performed by a very small number of specialist laboratories. In many cases, there is a clear need to develop assays with automated sample preparation modules incorporated. This would enable the delivery of an un-processed sample to the device, which in turn would enable measurement and clinical intervention close to real time and at the Point of Care (PoC).
A partially automated method for detecting and measuring propofol in biological samples, such as whole blood, has been described by McGaughran et al. in “Rapid measurement of blood propofol levels: A proof of concept study,” Journal of Clinical Monitoring and Computing, vol. 20, no. 5, pp. 381-381, 2006. The assay involves manual preparation of the blood sample, before introduction to an instrument that performs the remainder of the assay automatically. The assay is performed in the following way: First, a whole blood sample is osmotically lysed by dilution with deionised water at a ratio of 1:2. Then, the sample is transferred to a syringe and injected into the measurement device via a 1 μm syringe filter. The purpose of the filter is to mechanically lyse the remainder of the erythrocytes, erythrocyte ghosts (“empty” erythrocytes with an intact membrane) and large membrane fragments. It is essential to avoid injecting large cellular aggregates into the measurement system, which will potentially cause blockages and/or interfere with the extraction of the propofol from the sample. The automated portion of the assay involves delivery of a metered volume of the sample to a solid phase extraction (SPE) column, which extracts the propofol from the sample. After a series of washing steps, the propofol is eluted into the optical detection chamber using a metered volume of acetonitrile and reacted with a phenol-specific (Gibbs) reagent, namely 2,6 dichloroquinone-4-chloroimide, to produce a strongly coloured indophenol product. Absorption spectroscopy is used to measure the concentration of the coloured product, which is related to the original concentration of propofol in the whole blood sample.
The method described above has several advantages over the existing HPLC ones such as disclosed by G. F Plummer et al. and R. A. Uebel et al. For example, the device is much easier to use and maintain than the more general purpose HPLC instrument. Also, the device is significantly cheaper than HPLC and requires much smaller reagent volumes per measurement. Moreover, typical measurement times are much faster at approximately 3 to 5 minutes. Finally, the instrument has a much smaller footprint than the equivalent HPLC device, which is an important consideration for a busy clinical laboratory.
Unfortunately, several factors mean that the assay is unsuitable for routine clinical laboratory or PoC use. First, the lysis step requires accurate and precise metering of the sample and diluent volumes; this is a significant source of user error. Second, the extra blood manipulation presents an additional biohazard risk to the user. Third, as reported by A. L Dawidowicz et al. in “The advantages of cell lysis before blood sample preparation by extraction for HPLC propofol analysis,” Biomedical Chromatography, vol. 14, no. 7, pp. 493-497, 2000, as a significant (˜30-40%) proportion of the propofol is partitioned into cells retaining cell membranes on the syringe filter may result in a significant removal of the propofol from the sample before introduction to the instrument. This means that the final propofol concentration is not necessarily representative of the “true” concentration of propofol in the original whole blood sample. Overall, these factors mean the device is restricted to highly trained laboratory personnel in specialised laboratories. It is therefore desirable to incorporate the lysis (dilution and filtration) step into the instrument, so that the user can inject the blood directly into the device without prior sample preparation.
Despite the relative simplicity of the manual sample preparation protocol in the Gibbs/indophenol method for propofol detection, no methods or apparatus exist in the prior art which are compatible with automation of the lysis technique described above. In particular, handling small volumes of whole (rather than diluted) blood in a fluidic system is challenging. Reliable mixing of the blood with the lysing agent (water) in a defined ratio using narrow bore tubing is very difficult to achieve due to the variable viscosity of blood and the propensity of erythrocytes to stick to the walls of the tubing. The loss of erythrocytes is particularly problematic as a significant proportion of the drug (˜30-40%) is bound to these cells, thereby reducing the recovery by the SPE column. Also, the mixing of the blood with the lysing agent would typically require the use of two pumps which increases instrument complexity and cost. Incorporating an appropriate inline filter with enough capacity to perform the mechanical lysis step without blocking is difficult, as a large extra dead volume will be introduced into the system. This extra dead volume will lower the precision of the dispensed volumes, which in turn will introduce errors in the measured propofol concentration. Moving to harsher, detergent and/or acid-based lysis reagents such as disclosed in U.S. Pat. No. 7,247,484 would remove the erythrocyte ghosts and the larger membrane fragments and has the potential to remove the need for inline filtration entirely. However, these same detergents would interfere with the extraction of propofol by the reverse phase extraction column and produce errors.